Much has been learned about how the genetic instructions, written in an alphabet of just four 'letters' – the four different nucleotides in DNA – direct the formation of a bacterium, a fruit fly or a human. The information in RNA, although copied in a different chemical form, is still written in essentially the same language as in DNA: the language of a nucleotide sequence. Because U, like T, can form base pairs by hydrogen bonding with A (Figure 6–6), the complementary properties of base pairs described for DNA in Chapters 4 and 5 also apply to RNA (in RNA pairs G with C, and A pairs with U).
These promoters are characterized by two hexameric DNA sequences - the –35 sequence and the –10 sequence, named for their approximate location relative to the transcription start point (designated +1). The nucleotides shown in the figure are recognized by the σ factor, a subunit of the RNA polymerase holoenzyme. In humans, the CTD consists of 52 consecutive repeats of a Table 6-3 General Transcription Factors Required for Initiation of Transcription by Eukaryotic RNA Polymerase II.
Also Requires Activator, Mediator, and Chromatin- Modifying Proteins
Both ends of eukaryotic mRNAs are modified: by capping of the 5' end and by polyadenylation of the 3' end (Figure 6-21). The capping proteins only bind to the RNA polymerase tail when it is phosphorylated on Ser5 of the heptad repeat late in the process of transcription initiation (see Figure 6-15). The final cap contains a new 5'-to-5' linkage between the positively charged 7-methyl G residue and the 5' end of the RNA transcript (see Figure 6-21B).
The truncated 5' end of the intron becomes covalently linked to an adenine nucleotide as detailed in (B), creating a loop in the RNA molecule. The letter A highlighted in red forms the branching point of the lariat formed by the splice (see Figure 6–25). These snRNPs form the core of the spliceosome, a large assembly of RNA and protein molecules that carry out pre-mRNA splicing in the cell.
As transcription proceeds, the phosphorylated tail of RNA polymerase carries various components of the spliceosome (see Figure. Figure 6–29 One of the many rearrangements that occur in the spliceosome during pre-mRNA splicing. We have seen that the 5ʹ end of the pre-mRNA produced by RNA polymerase II is capped almost as soon as it emerges from the RNA polymerase.
The long C-terminal tail of the RNA polymerase coordinates these processes by transferring capping and splicing components directly to the RNA as it emerges from the enzyme. The position of the 3ʹ end of each mRNA molecule is specified by signals encoded in the genome (Figure 6–34). But of the pre-mRNA that is synthesized, only a small portion – the mature mRNA – is of further use to the cell.
Some of the proteins deposited on the mRNA while it is still in the nucleus can influence the fate of the RNA after it is transported to the cytosol. Almost half of the nucleotide sequences in this precursor rRNA are discarded and degraded in the core of the exosome. Unlike many of the major organelles in the cell, the nucleus is not bound by a membrane (Figure 6-42); instead, it is a huge aggregate.
FROM RNA TO PROTEIN
The amino acid corresponding to the codon/anticodon pair is attached to the 3' end of the tRNA. NET RESULT: AMINO ACID IS SELECTED BY ITS CODON C. Figure 6–55 The structure of the aminoacyl-tRNA linkage. The amino acid is linked to the nucleotide at the 3' end of the tRNA (see Figure 6–50). B) Actual structure corresponding to the boxed area in (A).
For other synthetases, the nucleotide sequence of the amino acid accepting arm (acceptor stem) is the most important recognition determinant. Some are free in the cytosol; others are attached to membranes of the endoplasmic reticulum. The figure shows the addition of the fourth amino acid (red) to the growing chain.
In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by breaking the high-energy bond between the tRNA and its amino acid). As indicated, the mRNA is translated in the 5'-to-3' direction and the N-terminal end of a protein is made first, with each cycle adding an amino acid to the C-terminus of the polypeptide chain (Movie 6.7 and Movies). 6.8). As explained in the text, EF-Tu allows proofreading of the codon-anticodon match.
Figure 6-67 Structure of the rRNAs in the large subunit of a bacterial ribosome, determined by X-ray crystallography. The proteins also help with the initial assembly of the rRNAs that make up the core of the ribosome. The rRNAs (5S and 23S) are shown in blue and the proteins of the large subunit in green.
The end of the protein-coding message is indicated by the presence of one of three stop codons (UAA, UAG, or UGA) (see Figure 6–48). As indicated, the translation of an mRNA sequence into an amino acid sequence on the ribosome is not the end of the process of forming a protein. The translation of the nucleotide sequence of an mRNA molecule into protein occurs in the cytosol on a large ribonucleoprotein assembly called a ribosome.
THE RNA WORLD AND THE ORIGINS OF LIFE
Each amino acid used for protein synthesis is first attached to a tRNA molecule that, by complementary base pair interactions, recognizes a particular set of three nucleotides (codons) in the mRNA. The mRNA molecule continues codon by codon through the ribosome in the 5'-to-3' direction until it reaches one of three stop codons. Eukaryotic and bacterial ribosomes are closely related, despite differences in the number and size of their rRNA and protein components.
In the final steps of protein synthesis, two different types of molecular chaperone control the folding of polypeptide chains. Much of our conclusions about the RNA world have come from experiments in which large pools of RNA molecules of random nucleotide sequences are generated in the laboratory. The cleavage, which occurs in nature at a distant site on the same RNA molecule that contains the ribozyme, is a step in the replication of the viroid genome.
Although not shown in the figure, the reaction requires a magnesium ion positioned at the active site. Although self-replicating systems of RNA molecules have not been found in nature, scientists have made significant progress in constructing them in the laboratory. It is plausible that this non-coded, primitive version of protein synthesis first evolved in the RNA world, where it would have been catalyzed by RNA molecules.
We know that ribozymes created in the laboratory can perform specific aminoacylation reactions; that is, they can match specific amino acids to specific tRNAs. It is therefore possible that tRNA-like adapters, each matching a specific amino acid, could have arisen in the RNA world, marking the beginning of a genetic code. If the evolutionary speculations embodied in the RNA world hypothesis are correct, early cells would have differed fundamentally from the cells we know today in that their hereditary information was stored in RNA rather than in DNA (Figure 6-93).
In the earliest cells, RNA molecules (or their close analogs) would have combined genetic, structural, and catalytic functions.
PROBLEMS
Ribose, like glucose and other simple carbohydrates, can be formed from formaldehyde (HCHO), a simple chemical that is easily produced in laboratory experiments that attempt to simulate conditions on primitive Earth. The sugar deoxyribose is more difficult to make and is produced in today's cells from ribose in a reaction catalyzed by a protein enzyme, suggesting that ribose precedes deoxyribose in cells. Presumably, DNA appeared on the scene later, but at the time proved more suitable than RNA as a permanent repository of genetic information.
In particular, the deoxyribose in its sugar-phosphate backbone makes chains of DNA chemically more stable than chains of RNA, so that much greater lengths of DNA can be maintained without breaking. The other differences between RNA and DNA—the double-helical structure of DNA and the use of thymine rather than uracil—further enhance DNA stability by making the many inevitable accidents that occur with the molecule much easier to repair, as detailed in chapter discusses 5 (pp. 271–273). From our knowledge of modern organisms and the molecules they contain, it seems likely that the development of the characteristic autocatalytic mechanisms that are fundamental to living systems began with the evolution of families of RNA molecules that can catalyze their own replication.
WHAT WE DON’T KNOW
6-8 The human α-tropomyosin gene is alternatively spliced to produce different forms of α-tropomyosin mRNA in different cell types (Figure Q6-3). For all forms of mRNA, the protein sequences encoded by exon 1 are the same, as are the protein sequences encoded by exon 10. One carries alanine and the other carries methionine at a site in the protein that normally contains valine (Figure Q6-4).
After treating these two mutants again with the mutagen, you isolate mutants of each that now carry threonine in place of the original valine (Figure Q6–4). Would you expect to be able to isolate valine-to-threonine mutants in one step. 6–10 Which of the following mutational changes would you predict to be most detrimental to gene function.
6–14 What is so special about RNA that it is believed to be an evolutionary precursor to DNA and protein. What is it about DNA that makes it a better material than RNA for storing genetic information. 6-15 If an RNA molecule could form a hairpin with a symmetrical inner loop, as shown in Figure Q6-5, the complement of this RNA could form a similar structure.
Random processes have just assembled a single copy of an RNA molecule with a catalytic site that can carry out RNA replication. This RNA molecule folds into a structure capable of joining nucleotides according to the instructions of an RNA template. Given a sufficient supply of nucleotides, this single RNA molecule will be able to use itself as a template to catalyze its own replication.
Figure V6–4 Two rounds of mutagenesis and the amino acids changed at a single position in a protein (Problem 6–9).
